1. Field of the Invention
The present invention relates to the purification of the procoagulant Factor VIII:C from source material such as plasma or cryoprecipitate which contains antihemophilic factor (herein, "AHF").
It is generally believed in this field that AHF in its natural form as obtained from plasma consists of aggregates of two molecular entities, which are termed Factor VIII:R and Factor VIII:C. Factor VIII:C is biologically active in correcting the coagulation defect of Hemophilia A. Factor VIII:R, also known as Factor VIII:WF (von Willebrand Factor), is biologically active in correcting the coagulation defect of von Willebrand's disease, a disorder of platelet aggregation. It is highly desirable to be able to purify Factor VIII:C with respect to Factor VIII:R and the other plasma proteins with which Factor VIII:C is normally found, including in particular fibronectin and fibrinogen.
2. Description of the Prior Art
Previously known processes for purifying Factor VIII:C introduce losses of yield and/or purity which up to now have been tolerated. The present invention achieves higher levels of purification and yield, without deactivation, in a manner which is not suggested by the prior art.
D. E. G. Austen, "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose", in British Journal of Hematology, 1979, (43) 669-674, described a chromatographic separation process in which human or porcine Factor VIII concentrate was passed through a column of 6-amino-n-hexyl-substituted agarose. The column and all eluting solutions were at a pH of 5.5. A high degree of separation of Factor VIII:C from Factor VIII:R, and a high degree of purification of Factor VIII:C from other proteins, were obtained. However, the total recovery of human Factor VIII:C was only 35-40%, and for porcine Factor VIII:C was only 24-30%. The authors indicate that more acidic pH values in the buffers (down to a pH of about 5.2) favor higher purification of Factor VIII, and they purposely chose the pH of 5.5 in order to have as acidic an environment as possible without suffering too low a yield. Thus, the authors teach away from higher (that is, less acidic) pH values.
Several more recent publications have continued to insist on maintaining an acid pH in the chromatographic column. Morgenthaler, "Chromatography of Antihemophilic Factor on Diaminoalkane- and Aminoalkane-Derivatized Sepharose", Thromb. Haemostas. 47(2) 124-127 (1982), found that when AHF was chromatographed on Sepharose CL-2B agarose gel at pH values of 6.0, 6.5 and 7.0, no significant separation of Factors VIII:C and VIII:R could be obtained. Chromatography of AHF at a pH of 5.5 produced a very marked separation between Factors VIII:C and VIII:R. An even more recent paper, by Faure et al., Note, "Improved buffer for the chromatographic separation of Factor VIII coagulant," J. Chromatography 257 (1983), 387-391, retains the pH value of 5.5 indicated by Austen and attempts to improve the performance of that chromatographic procedure by adding compounds to the buffers. And Austen, in an attempt to improve the results obtained in his article discussed above, continues to operate at a pH of 5.5 and achieves a yield of Factor VIII:C of about 40%, in Austen et al., "Factor VIII Fractionation on Aminohexyl Sepharose with Possible Reduction in Hepatitis B Antigen", Thromb. Haemostasis, 48(1), 46-48 (1982).
To our knowledge, Factor VIII:C has been applied to a column at a pH closer to neutral only in the instance in which Factor VIII:R and a large number of other contaminants, including fibronectin and fibrinogen, have already been separated from the Factor VIII:C. Specifically, in U.S. Pat. No. 4,361,509, Zimmerman et al. employ a column bearing monoclonal antibodies to Factor VIII:R to recover a dilute solution of Factor VIII:C that has been ultrapurified of Factor VIII:R. The solution of ultrapurified Factor VIII:C is concentrated by adjusting the pH to 6.8 with buffer, applying the solution to a column of aminohexyl agarose, and then eluting the Factor VIII:C from the column. This concentration step starts from material which is about 1000 times as pure as the starting material, and thus does not suggest what the results would be if substantial amounts of VIII:R were present; indeed, if one who had read this patent were presented with source material containing both Factors VIII:C and VIII:R one would employ the extremely effective monoclonal antibody-column separatory technique to remove the Factor VIII:R. Thus, the teaching of the U.S. Pat. No. 4,361,509 patent regarding the conditions for employing the aminohexyl agarose column does not contradict or modify the teachings of the articles discussed above.